EP3240897B1 - Mehrschichtiger komplex, verfahren zur herstellung dieses komplexes und verwendung dieses komplexes - Google Patents

Mehrschichtiger komplex, verfahren zur herstellung dieses komplexes und verwendung dieses komplexes Download PDF

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EP3240897B1
EP3240897B1 EP15823699.2A EP15823699A EP3240897B1 EP 3240897 B1 EP3240897 B1 EP 3240897B1 EP 15823699 A EP15823699 A EP 15823699A EP 3240897 B1 EP3240897 B1 EP 3240897B1
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compound
layer
inorganic silicate
magnetic
nucleic acids
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French (fr)
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EP3240897A1 (de
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Arnaud Burr
Ali Laayoun
Alain Laurent
Raphäel VEYRET
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Biomerieux SA
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Biomerieux SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B5/00—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts
    • B32B5/02—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by structural features of a fibrous or filamentary layer
    • B32B5/08—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by structural features of a fibrous or filamentary layer the fibres or filaments of a layer being of different substances, e.g. conjugate fibres, mixture of different fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B9/00—Layered products comprising a layer of a particular substance not covered by groups B32B11/00 - B32B29/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2262/00—Composition or structural features of fibres which form a fibrous or filamentary layer or are present as additives
    • B32B2262/10—Inorganic fibres

Definitions

  • This disclosure relates to the field of molecular diagnostics. More specifically, it relates to solid magnetic complexes allowing the extraction and/or purification of nucleic acids (DNA and/or RNA) from biological samples or environmental samples, these complexes being provided with at least three distinct layers .
  • nucleic acids DNA and/or RNA
  • sample Prep for sample preparation: preparation of the sample
  • nucleic acid extraction is generally critical because from this first step will result all the quality of a series of events leading to the final result of the diagnostic test. Indeed, it is necessary to have nucleic acid extraction as specific and efficient (in quantity, purity and time) as possible in order not to lose information, which can lead to an erroneous diagnosis and be fatal for a patient.
  • Newer nucleic acid extraction techniques use solid phases where cells are lysed under specific reaction conditions and the released nucleic acids bind to the solid phase. It is well known in the state of the art that current techniques for extracting nucleic acids very often use solid phases which are particles coated with silica. Indeed, silica has the property of reversibly adsorbing nucleic acids under certain conditions of salt concentration and pH, which makes it a very suitable material for this use. These techniques are clearly described in “Rapid and simple method for purification of nucleic acids. » Boom, Journal of Clinical Microbiology, 1990 p495 and in the patent US 5,234,809 from the same author.
  • magnetic silica particles can be very effective in the extraction of nucleic acids, their manufacturing protocol is long, delicate and expensive. Indeed, the magnetic particles coated with silica used for the extraction nucleic acids can sometimes be very complex to manufacture, particularly at the level of the silica layer which needs to be perfectly stable and controlled in thickness and homogeneity. The quality and nature of this silica layer is of fundamental importance for the quality of the extracted nucleic acids as well as for the reproducibility of the results from one extraction of the nucleic acids to another.
  • US 2010/0009375 A1 describes the manufacture of magnetic particles coated with a continuous and ultrathin layer of silica less than 1 nm for the extraction of nucleic acids.
  • This layer is however very difficult to measure and remains difficult to control at the process level. This makes the quality control of these particles very complex. If the silica layer is too thin, the nucleic acids can be adsorbed on the magnetite which makes up the magnetic core without being able to be desorbed during the elution step.
  • the manufacturing process is very complex and involves strict control of the SiO 2 /magnetite/Na 2 O reagent ratios.
  • US 2011/0186524 A1 describes the manufacture of magnetic silica particles for the extraction of nucleic acids, involving a heating step at 200°C for 7 hours and the use of organic solvents, all of which have drawbacks in terms of preparation time, cost and subsequent use (amplification, detection) of the extracted nucleic acids because, depending on the treatments received during the extraction, the amplification and detection yields may be affected.
  • US2009/182120 describes a multi-layered complex consisting of magnetic particles and silica nanoparticles for the extraction of nucleic acids.
  • metal oxide particles by grafting various organic or inorganic compounds thereto.
  • said particles are stabilized by complexation with derivatives of phosphates, phosphonates, carboxylates or other organic compounds. This is described in the book “Stability constants of metal-ioncomplexes” by Lars Gunnar Séen and Arthur Earl Martell published in 1971 by the Chemical Society , in US 5160725 , by G. Pourroy in Chem. Com. 2010 46 985-987 or in Chem. Mater., 2008, 20 (18), pp 5869-5875 .
  • These complexes can be used as contrast agents in medical imaging, as a probe to detect a complementary DNA strand in vivo or as biocompatible ferrofluids.
  • the object of this disclosure relates to the development and the production of new solid complexes allowing the extraction and the purification of nucleic acids in an efficient manner and this from samples, preferably from complex biological samples.
  • the present invention also relates to the use of this three-layered complex in the purification of microorganisms and/or biomolecules or in the extraction of biomolecules, preferably nucleic acids from a sample.
  • the present invention relates to the use of this three-layer complex in a method for lysing microorganisms and/or cells, from a sample, characterized in that it consists in bringing at least one sample into contact with at least one complex according to the invention and in which the said at least one inorganic silicate compound and/or the said at least one compound having an affinity for the said at least at least one magnetic compound and/or for said at least one inorganic silicate compound comprises at least one agent from the family of detergents allowing lysis.
  • the present invention relates to molecular diagnostic kits comprising at least the three-layer complex according to the invention.
  • layer is meant a thickness of material or of a substance extended so as to form a film of variable thickness.
  • the layer can be continuous or discontinuous.
  • the layer can also be called a coating.
  • three-layer complex is meant a solid and compact assembly consisting of at least three physically and chemically distinct layers.
  • the layers overlap, the first layer is covered by the second which matches the shape of the first layer, and the second layer is itself covered by the third and so on.
  • the succession of the three layers is done in a precise order to observe the properties of the complex.
  • the second layer is discontinuous, the third layer overlaps and is in direct contact with both the second layer and the first layer.
  • the first layer cannot overlap the second or the third layer, just as the second layer cannot overlap the third layer.
  • magnetic compound By magnetic compound is meant a compound capable of reacting to a magnetic field by an orientation and/or displacement reaction dependent on the strength and orientation of said magnetic field. This force takes place through the magnetic field, and is produced by moving charges or magnets.
  • the magnetic compounds included in the three-layer complex according to the invention are chosen from metals and/or metal oxides.
  • the metal or metals endowed with magnetic capacities which can be used in the three-layer complex of the present invention are iron, cobalt, nickel, steels, cast iron as well as metals which react more weakly to magnetism such as manganese, chromium, platinum and aluminum.
  • the magnetic compound of the complex of the invention is magnetite or maghemite, even more preferably magnetite.
  • Said at least one magnetic compound of the complex of the invention can be in planar form and form a membrane, a plate. It can also be in variable form, for example in cubic form, in the form of a crystal, of a needle, of a cone, of a sphere, in the form of a particle which can itself be spherical, multihedral, for example tetrahedral, octahedral ...
  • the preferred form of the magnetic compound of the tri-layer complex of the invention is the particle of size between 1 and 500 nm, preferably between 2 and 400 nm, more preferably between 10 and 300 nm, more preferably still between 50 and 150nm.
  • particle an identifiable physical structure, distinct, isolated and not soluble in an aqueous medium or a mixture of organic and aqueous solvents or in an organic solvent and whose size can be micrometric or nanometric.
  • the particle is nanometric and between 1 and 400 nm.
  • said at least one magnetic compound of the tri-layer complex is in the form of a particle, then the whole of the tri-layer complex has the shape of a particle because the layer of silicate inorganic compound covering said at least one magnetic compound takes the shape of the latter and the same is true for the third layer against the second layer.
  • said at least one magnetic compound forms the core of said particle since it is placed at the center thereof.
  • the final shape of the three-layer complex is not necessarily identical to the initial shape of said at least one magnetic compound.
  • the three-layer complex has the shape of a blackberry, the magnetic compound constituting the solid core and the second layer forming a discontinuous layer formed by satellite nano-spheres around the core.
  • the layer of magnetic compound has a micrometric or nanometric thickness.
  • inorganic silicate compound any compound comprising silica and being of inorganic nature.
  • the inorganic silicate compound(s) that can be used in the three-layer complex according to the invention are silicates, magnesium, sodium, potassium, lithium or calcium silicates, talc, alumino-silicates, kaolin, bentonite, silica nanoparticles, preferably silica nanoparticles whose size is between 0.1 and 20 nm, preferably between 1 and 20 nm, mesoporous silica nanoparticles, magnetic nanoparticles coated with silica, as well as the nanoparticles mentioned above, the silica of which is chemically modified with organic or inorganic groups.
  • the organic or inorganic groups which can modify the silica are compounds having carbon groups endowed with amine, carboxylic, thiol, alcohol, phosphonic or sulphonic acid, phosphonate and/or phosphate functions, compounds of the detergent family such as saponins, homopolymers or copolymers, polymers of maleic anhydride, N-vinylpyrrolidone, polyethylenes, propylene and methyl vinyl ethers (MVE) grafted with maleic anhydride, N-vinylpyrrolidone (NVP)/N- acryloxysuccinimide (NAS), polysaccharides, amino latexes.
  • MVE methyl vinyl ethers
  • the inorganic silicate compound is bentonite or silica nanoparticles whose size is between 0.1 and 20 nm, even more preferably between 1 and 20 nm. These compounds give the best results in terms of nucleic acid extraction.
  • the inorganic silicate compound consists of silica nanoparticles, the silica of which is modified by bonding with saponins.
  • the bond between the first layer of the complex of the invention comprising at least one magnetic compound and the second layer comprising at least one inorganic silicate compound is made by electrostatic or covalent bonds, giving rise to a three-dimensional structure.
  • said at least one inorganic silicate compound is in particulate form such as a grain, a sheet, a needle, a fiber, a nanoparticle, etc. form insoluble in an aqueous solvent, an aqueous solvent-organic solvent mixture or an organic solvent.
  • silica can be found in grain or particle form
  • bentonite is generally found in sheet form.
  • the particles or forms which are not soluble in an aqueous solvent or an aqueous solvent-organic solvent mixture or an organic solvent of the inorganic silicate compound generally have a nanometric size to allow good adhesion of the at least one inorganic silicate compound to the layer of at least one magnetic compound.
  • the size of these non-soluble particles or forms is between 0.1 and 20 nm, preferably between 1 and 20 nm, more preferably between 2 and 10 nm, even more preferably between 6 and 8 nm.
  • the preferred particulate form of the inorganic silicate compound is the nano-particulate form.
  • the first layer of the three-layer complex is coated, at least partially, with a second layer consisting of silica nano-particles, preferably from 0.1 to 20 nm, more preferably between 1 and 20 nm, more preferably again between 2 and 10 nm and even more preferably between 6 and 8nm.
  • the silica nanoparticles marketed by the company Sigma-Aldrich, called Ludox ® are used as the inorganic silicate compound. Ludox ® SM 7 nm nanoparticles are very particularly preferred.
  • form not soluble in an aqueous solvent or aqueous solvent-organic solvent mixture or in an organic solvent is meant a structure which does not dissolve or only partially dissolves in these solvents, whatever the temperature of the medium.
  • this form remains well materialized, in the form of a suspension, structured and identifiable. It can be observed under an electron microscope.
  • the figure 3 illustrates magnetite particles (magnetic compound) covered with 7 nm Ludox SM silica nanoparticles (silicated inorganic compound), themselves covered with pyrophosphate (compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound).
  • This figure thus exemplifies the complex of the invention with the inorganic silicate compound in the insoluble form in an aqueous solvent, in an aqueous solvent-organic solvent mixture or in an organic solvent.
  • the layer of inorganic silicate compound has a nanometric thickness.
  • compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound is meant a compound capable of binding to the magnetic compound or capable of binding to the inorganic silicate compound, whatever their forms. respective. More specifically, they are molecules with an affinity for metal oxides as described in "Stability constants of metal-ion complexes" by Lars Gunnar Séen and Arthur Earl Martell published in 1971 by the Chemical Society".
  • citric acid and its salts phosphate, pyrophosphate, triphosphate, polyphosphate ions, phosphonates and phosphonic acids, phosphonates or phosphonic acids coupled to organic molecules , compounds from the family of phosphoric acids, sulfonates, compounds from the family of detergents and/or compounds from the family of carboxylic acids.
  • the organic molecules coupled to the phosphonates or phosphonic acids can be, for example, riboses, deoxyriboses, amino acids, peptides, etc.
  • riboses for example, riboses, deoxyriboses, amino acids, peptides, etc.
  • saponins Tweens, Tritons, ...
  • a three-layer complex according to the invention the third layer of which consists of a combination of compounds having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound mentioned above.
  • phosphate, pyrophosphate or triphosphate ions in combination with a saponin.
  • said at least compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound is a compound having an affinity for iron.
  • said at least one compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound consists of monophosphates, pyrophosphates, triphosphates, citric acid or its salts and/or saponin.
  • the bond between the second layer of the complex of the invention comprising at least one inorganic silicate compound and the third layer comprising at least one compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound made by electrostatic or covalent or coordination bonds giving rise to a three-dimensional structure.
  • the second layer of the complex according to the invention consists of particles or nanoparticles, preferably silica nanoparticles
  • interstices can occur between said particles or nanoparticles and the third layer comprising at least one compound having an affinity for at least one magnetic compound adheres more easily and better to the first layer because some metal atoms and/or metal oxides of the first layer are available and more accessible to form a reaction or effect a bond
  • the layer of compound having an affinity for the at least one magnetic compound and/or the at least one inorganic silicate compound has a nanometric thickness or less.
  • the three-layer complex according to the invention and as defined above also comprises a solid support under all or part of the first layer constituted by the at least one magnetic compound.
  • solid support any support capable of supporting the layer of at least one magnetic material. It can be at least one flat support, a hollow support, a wafer, a needle, a membrane, a plate, a sheet, a cone, a tube, a ball, a particle...
  • the support is preferably a ball or a particle.
  • the preferred form is a three-layer complex having the form of a particle, the first layer of which constitutes the core of said particle and has a size comprised between 2 and 400 nm, preferably between 50 and 100 nm.
  • the most particularly preferred forms of the complex according to the invention are complexes comprising magnetite between 50 nm and 100 nm (first layer), silica nanoparticles (Ludox SM 7 nm most preferably) or bentonite (second layer) and monophosphate ions , pyrophosphates, triphosphates (third layer).
  • the ratio by weight per weight (or w/w) between said at least one inorganic silicate compound and said at least one magnetic compound represents between 0.1% and 60%, preferably between 1% and 50%, more preferably between 3% and 35%, more preferably still between 4 and 10%.
  • the ratio by weight/weight of inorganic silicate compound to magnetic compound is higher when the inorganic silicate compound is in the form of a sheet, such as for example bentonite, than when it is in form of nanoparticles.
  • the bentonite/magnetite ratio is between 25% and 55%, whereas if it is covered with Ludox nanoparticles, the Ludox/magnetite ratio is between 1% and 10%.
  • the molar ratio of said at least one magnetic compound (preferably iron)/at least one compound having an affinity for said at least one inorganic silicate compound and/ or at least one magnetic compound is between 0.1% and 15%, preferably between 0.1 and 10%, more preferably between 5% and 10% and even more preferably between 6% and 9%.
  • the three-layer complex according to the invention is produced by manufacturing methods that are easy to implement and highly reproducible. This makes it possible to have reliability in the performance of the complex produced as well as uniformity, or at least very little variation, from one batch of complex produced to another.
  • Said at least one magnetic compound is either found in the natural state, or it is synthesized according to standard protocols described in the literature or by coprecipitation as described by R. Massart (IEEE Trans. Magn. 1981, 17, p1247-1248 .), or by partial oxidation of metal salts as described by T. Sugimoto and E. Matjevic (Journal of Colloids and Interface Science, 1980, 74, P227-243 ), or by decomposition of organometallic precursors as described by Maity (Journal of Magnetic Materials 321 1256 (2009 )).
  • the advantage of these techniques is that one can easily incorporate other metal atoms such as cobalt, manganese, zinc, etc. so as to obtain ferrites which can also be used as a magnetic compound as described above in the complex of the invention.
  • said at least one magnetic compound can be purchased commercially. This is particularly the case for nanoparticles of metal oxides which can be purchased from suppliers of pigments for magnetic inks as described in The Journal of Imaging Science and Technology November/December 2000, vol. 44, no. 6; p. 508-513 "The Influence of Particle Size, Shape and Particle Size Distribution on Properties of Magnetites for the Production of Toners ").
  • Said at least one inorganic silicate compound as described above for example of the magnesium or sodium trisilicate type, bentonite, kaolin, talc, etc. can be purchased easily from chemical product suppliers such as Sigma-Aldrich (Saint Louis, USA ). Furthermore, many standard protocols known to those skilled in the art describe how to obtain these inorganic silicate compounds.
  • the inorganic silicate nanoparticles, used in the preferred embodiment of the invention can be synthesized according to standard protocols for the condensation of tetraethoxy silane (TEOS) in aqueous and alkaline medium which are described in the literature by W. Stöber (Journal of Colloids and Interface Science 1968, 26, p62-69 ).
  • TEOS tetraethoxy silane
  • the silicate nanoparticles can be obtained from suppliers of chemical products such as the company Sigma-Aldrich (Saint Louis, USA.) supplying, for example, the particles of the LUDOX range or equivalents thereof or such as the NanoH company (Lyon, France). It is possible and easy to introduce functional groups such as amino, phosphonates, azido, alkyne, etc. onto these inorganic silicate compounds (preferably nanoparticles) by co-condensation of the corresponding silanes with TEOS so as to increase the force of interaction with the at least one magnetic compound (for example phosphonate nanoparticles).
  • suppliers of chemical products such as the company Sigma-Aldrich (Saint Louis, USA.) supplying, for example, the particles of the LUDOX range or equivalents thereof or such as the NanoH company (Lyon, France). It is possible and easy to introduce functional groups such as amino, phosphonates, azido, alkyne, etc. onto these inorganic silicate compounds (preferably nanoparticles)
  • one or more washes in water or in an aqueous medium are carried out after each of the steps for producing the complex according to the invention, preferably after step a) and b).
  • the bonding of the second layer to the first layer takes place by adsorption and by electrostatic bonding and/or by covalent bonding and/or a coordination bond. The same goes for bonding the third layer to the second layer.
  • the backing is millimeter to micrometer thick.
  • a material making it possible to physically or chemically fix the first layer of the complex to the support can be an adhesive or any other compound capable of fulfilling this function, namely an intermediate substance capable of maintaining the bond between the solid support and the compound magnetic such as, for example, a protein, a polymer, a hydrophilic or hydrophobic polymer, a polydopamine, etc...
  • the final complex is in the form of a particle comprising a core of at least one magnetic compound, which is covered at least partially with two layers: a layer of at least one inorganic silicate compound and a layer of compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound, this latter layer being located outside the complex.
  • the interaction between said at least one magnetic compound and said at least one inorganic silicate compound is non-covalent, likewise for the interaction between said at least one compound having an affinity for said at least one compound silicate inorganic compound and/or said at least one magnetic compound and said at least one silicate inorganic compound or said at least one magnetic compound.
  • the contact between said at least one magnetic compound and said at least one inorganic silicate compound generally takes place at a pH of between 2 and 7, preferably between 3 and 6, most preferably between 3 and 4.
  • the coating of the first layer of said at least one magnetic material is favored by opposing electrostatic interactions.
  • the coating must be made in a pH zone where the at least silicate inorganic compound and said at least one magnetic compound have opposite surface charges as described US 4280918 .
  • magnetite is used as the magnetic compound and silica is used as the inorganic silicate compound.
  • silica is used as the inorganic silicate compound.
  • the isoelectric point of magnetite being at 6.8 and that of silica around 2, the coating of magnetite with silica must be carried out at a pH between 2 and 6.8, preferably at pH 3 ,5.
  • the temperatures used are between 15° C. and 65° C., preferably between 20° C. and 60° C. used.
  • the preferred embodiment of the invention is very easy and quick to synthesize because it uses commercial or easily synthesized compounds.
  • said at least one magnetic compound preferably magnetite particles
  • said at least one silicate inorganic compound preferably Ludox silicate inorganic nanoparticles
  • the two types of compounds bind together in a very solid way by forming a three-dimensional structure (in the shape of a blackberry in the case of the use of magnetite and Ludox particles).
  • the temperature used in this first step is room temperature but the temperature can vary between 20°C and 60°C.
  • the composite complexes obtained that is to say the two-layer complexes (or in the preferential form the particles covered with Ludox) are then washed by magnetization or by any known appropriate technique making it possible to obtain the same function.
  • a second step we coat at least one compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound on the two-layer complex consisting of at least one magnetic compound covered with at least one inorganic silicate compound. This is done by incubating said two-layer complex with said at least one compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound in an aqueous medium for 30 seconds to 24 hours, preferably 2 hours, at controlled pH and temperature.
  • the temperature used at this stage is room temperature but the temperature can vary between 20°C and -60°C.
  • the three-layer complex is again washed by magnetization or any other known technique having the same function, then it can be taken up in water in order to determine the concentration by measuring the dry extract.
  • the washings are generally carried out in water or at least in an aqueous medium.
  • one of the particularly advantageous methods of manufacturing the complex is to implement at least one magnetic compound, preferably at least one particle of magnetite or maghemite, and to deposit on the latter or to at least partially covering the latter with at least one inorganic silicate compound, preferably an inorganic silicate compound of insoluble nature and already structured as particles (for example silica nanoparticles) or sheets (for example bentonite).
  • inorganic silicate compound use will preferably be made of silica nanoparticles between 0.1 and 20 nm, more preferably between 1 nm and 20 nm and even more preferably 7 nm Ludox® SM particles.
  • this embodiment of the complex implies an increase in the developed surface of the complex (or specific surface) and therefore an increase in the capacity for extraction/purification of nucleic acids.
  • the magnetic compound can easily be functionalized with these functions without the need to involve complex processes to control of condensation of silanes on magnetite in the presence of organic solvent(s).
  • said at least one magnetic compound In order to offer very good capacities for capturing and extracting and/or purifying nucleic acids, it is preferable for said at least one magnetic compound to be completely coated with said at least one inorganic silicate compound.
  • the complex according to the invention offers remarkable and particularly advantageous properties in the capture and elution of nucleic acids and in particular in complex biological media or samples.
  • the three-layer complexes of the invention have been evaluated for their ability to extract nucleic acids from cells or bacteria contained in a complex medium or sample such as blood.
  • sample is meant a sample having various origins such as samples of food, environmental, human, veterinary or cosmetic origin.
  • samples of food origin mention may be made, in a non-exhaustive manner, of a sample of milk products (yogurts, cheeses, etc.), meat, fish, eggs, fruit, vegetables, beverages (milk, fruit juice, soda, etc.).
  • these samples of food origin can also come from sauces or more elaborate dishes or unprocessed or partially processed raw materials.
  • a food sample can also come from a food intended for animals, such as cakes, animal meal. All these samples, if they are not liquid, are previously treated to be in liquid form.
  • the sample may be of environmental origin and may consist, for example, of a surface, water, etc. sample.
  • the sample may also consist of a biological sample, of human or animal origin, which may correspond to samples of biological fluid (urine, whole blood or derivatives such as serum or plasma, sputum or saliva, pus, cerebrospinal fluid, etc. ), stool (e.g. cholera diarrhea), swabs of nose, throat, skin, wounds, organs, tissues or isolated cells, swab specimens, bronchoalveolar swabs or washes , biopsies.
  • biological fluid urine, whole blood or derivatives such as serum or plasma, sputum or saliva, pus, cerebrospinal fluid, etc.
  • stool e.g. cholera diarrhea
  • swabs of nose, throat, skin, wounds, organs, tissues or isolated cells swab specimens, bronchoalveolar swabs or washes , biopsies. This list is obviously not exhaustive.
  • sample refers to a part or quantity, more particularly a small part or quantity, taken from one or more entities for the purpose of analysis.
  • This sample may optionally have undergone a prior treatment, involving for example steps of mixing, dilution or even grinding, in particular if the starting entity is in the solid state.
  • the sample analyzed is, in general, likely to - or suspected of - containing at least one biomolecule representative of the presence of microorganisms or of a disease to be detected, characterized or monitored.
  • biomolecule we mean a compound or a chemical entity which can be a nucleic acid (DNA or RNA of any type genomic DNA, complementary DNA, messenger RNA, complementary RNA, transfer RNA, mitochondrial RNA, chloroplast DNA, ribosomal RNA, DNA plasmid, viral DNA or RNA, microRNA, snoRNA, siRNA, RNAi, in single-stranded or double-stranded form) or a protein.
  • DNA or RNA of any type genomic DNA, complementary DNA, messenger RNA, complementary RNA, transfer RNA, mitochondrial RNA, chloroplast DNA, ribosomal RNA, DNA plasmid, viral DNA or RNA, microRNA, snoRNA, siRNA, RNAi, in single-stranded or double-stranded form or a protein.
  • microorganism all or part of a bacterium, a fungus, a yeast or a virus.
  • the present invention also relates to a method for purifying microorganisms and/or biomolecules or for extracting biomolecules, preferably nucleic acids, from a sample, preferably biological, in which at least a complex as defined above.
  • the extraction within the meaning of the invention includes the lysis and the purification of the biomolecules.
  • the purification itself comprises the adsorption or capture, the washing and the elution of the biomolecules and/or the microorganisms.
  • Capture consists in adsorbing the biomolecules and/or microorganisms on the complex and elution upon desorption or release of the latter from the complex according to the invention.
  • the tests of the complex are carried out under chaotropic conditions which are conditions where the three-dimensional structures of biological macromolecules, such as proteins, DNA or RNA are denatured.
  • Chaotropic agents interfere with weak (non-covalent) intramolecular interactions, such as hydrogen bonds, van der Waals forces and hydrophobic forces.
  • chaotropic agents mention may be made of urea, guanidine salts such as guanidinium chloride or thiocyanate and lithium perchlorate. They are generally used in concentration ranging from 1 to 6 M, in particular for GuSCN and GuHCl.
  • a detergent is also added which helps cell lysis, the latter can be chosen from Tween, tritons, SDS and other commonly used detergents at concentrations between 0.05 and 5% by weight or by volume per compared to lysis buffer,
  • proteins capable of denaturing or damaging nucleic acids such as nucleases are inhibited or destroyed under chaotropic conditions and this offers the most favorable conditions for efficiently extracting nucleic acids.
  • guanidinium chloride or guanidinium thiocyanate and/or hydrochloric acid (HCl) in a buffered medium at pH 7 and a detergent, preferably Triton X100.
  • the complex according to the invention offers high performance properties in the purification and/or the extraction of biomolecules and/or microorganisms and in particular in the extraction of nucleic acids.
  • said at least one compound having an affinity for said at least one inorganic silicate compound and/or for the at least magnetic compound completely displaces the coating of inorganic silicate compound and causes the DNA extraction yield to drop .
  • the tri-layered complex would improve RNA elution capabilities and thus offer significantly improved DNA and RNA nucleic acid purification and extraction properties. compared to the prior art.
  • the particularly preferred form of inorganic silicate compound consists of silica nanoparticles between 0.1 and 20 nm, more preferably between 1 and 20 nm. It should be noted that the larger the size of the nanoparticles of silicate inorganic compound bound to the first layer -layer magnetic- of the complex of the invention is small, the more the extraction of the nucleic acids with the complexes according to the invention is important and effective.
  • the particle size influences the extraction yield of nucleic acids and the greater the specific surface of the nanoparticles of silicate inorganic compound, the better the extraction of nucleic acids: a complex having magnetite covered by nanoparticles of inorganic silicate compound Ludox ® HS 40 will capture fewer nucleic acids than a complex having magnetite covered by nanoparticles of inorganic silicate compound Ludox ® SM 7nm.
  • the ratios by weight by weight of inorganic silicate compound/magnetic compound are between 0.1% and 60%, preferably between 0.5% and 30%, more preferably between 1% and 20%, more preferably still between 2% and 15%, very particularly preferably between 3% and 10%.
  • the ratios by weight by weight of magnetic compound/compound having an affinity for said at least one compound magnetic and/or for said at least one inorganic silicate compound are between 0.1% and 20%, preferably between 0.1% and 10%., more. preferably between 4% and 8%.
  • biomolecules have been extracted with the complex according to the invention, it is advantageous to detect the target nucleic acids, or even to quantify them in order to make an accurate diagnosis.
  • target nucleic acids are those suspected or likely to be present in the sample tested.
  • the conventional detection methods that can be used are all those widely known to those skilled in the art.
  • radioactive markings cold markings: colorimetry, fluorescence, chemiluminescence; molecular hybridizations; Southern Blots; Northern Blots; Dot Blots or in situ hybridization.
  • detection is used with a detection probe.
  • detection probe or probe is meant a nucleic sequence of a nucleotide sequence of four bases of different types chosen from the group of adenine, thymine, guanine, uracil, cytosine, which is capable of hybridizing specifically on an amplicon and comprising at least one marker.
  • the probe can be a rounded probe (called O-probe, see Applicant's patent application FR08/54549 filed July 4, 2008 ), a molecular beacon (otherwise called Molecular Beacon), a Taqman ® probe or a FRET probe. These last three types of probes are well known to those skilled in the art. These probes may optionally consist wholly or partly of modified nucleotides.
  • Each probe comprises a label and optionally a quencher.
  • probes emitting fluorescence when they hybridize to the complementary sequence probes described by Tyagi & Kramer (Nature Biotech, 1996, 14:303-308 ), commonly known as “molecular beacons” or commercial kits such as, for example, kits from the R-gene ® range from Argene (bioMérieux, Verniolle, France). It is possible to do the detection in real time or at the end of the reaction.
  • quantification methods used are the standard quantification methods conventionally used by those skilled in the art. For example, it is possible to use quantification kits from the R-gene ® range from Argene (bioMérieux, Verniolle, France). These methods conventionally involve standard quantification ranges (QS) making it possible to evaluate the actual quantity of a given nucleic acid or nucleic acids in the sample tested.
  • QS standard quantification ranges
  • the present invention relates to a method for detecting and/or quantifying target nucleic acids as described above, in which between the extraction step and the nucleic acid detection step, there there is a step for eluting the nucleic acids of the complex implemented in the step extraction and / or a step of amplification of nucleic acids by conventional techniques.
  • the elution step is generally carried out using a solution with a slightly alkaline pH and low ionic strength at a temperature between 50°C and 70°C.
  • a solution with a slightly alkaline pH and low ionic strength at a temperature between 50°C and 70°C.
  • commercial products such as the products of the NucliSENS easyMAG extraction range (bioMerieux, France). These conditions are standard conditions for maintaining the integrity of nucleic acids and are standard conditions.
  • amplification or amplification reaction is meant any nucleic acid amplification technique well known to those skilled in the art.
  • the amplification step is generally done because the quantity of nucleic acids to be detected and/or quantified is very low and it is necessary to go through an amplification phase of the latter to detect and/or quantify them in order to to give an accurate diagnosis. Without an amplification phase, it is entirely possible to obtain erroneous results concluding that a target nucleic acid is absent from a sample when in reality it is present in the latter, but in such a small quantity that the techniques used do not make it possible to detect it.
  • many amplification methods can be used such as PCR, RT-PCR, LCR, RCR, 3SR, RCA, NASBA, TMA, SDA or any nucleic acid amplification techniques known to those skilled in the art.
  • the present invention relates to a method for lysing microorganisms and/or cells and/or tissues, from a sample, characterized in that it consists in bringing into contact at least a sample with at least one three-layer complex as described above and in which said at least one inorganic silicate compound and/or said at least one compound having an affinity for said at least one magnetic compound and/or for said at least one least one inorganic silicate compound comprises at least one agent from the family of detergents allowing lysis.
  • lyse cells the cells of a tissue and/or microorganisms in a targeted manner.
  • the use of this complex can be interesting when it comes to targeting a certain type of cell or microorganism.
  • the membranes and nucleic acids of human blood cells present in large quantities in a sample. This can be achieved with saponin which penetrates and weakens membranes and envelopes containing cholesterol. It may be necessary to carry out this selective lysis in order to be able to select and extract the pathogenic microorganisms present in very small quantities in the sample tested.
  • the compounds constituting the second and/or the third layer of the complex of the invention can be functionalized with compounds from the family of detergents, such as saponins for example.
  • these complexes thus functionalized may have a role in the selective lysis of human cells.
  • the three-layer complex could be used to carry out a first stage of lysis and selective capture of non-targeted nucleic acids and to facilitate in a way a stage of targeted enrichment of microorganisms present in the sample but not captured by the complexes of the invention. It can also be envisaged to have a three-layer complex according to the invention of which said at least one inorganic silicate compound and/or said at least one compound having an affinity for said at least one magnetic compound and/or for said at least an inorganic silicate compound are functionalized in different and complementary ways and make it possible to capture and/or lyse different types of biomolecules and/or microorganisms.
  • said at least one inorganic silicate compound comprises nanoparticles of silica between 0.1 nm and 20 nm, more preferably between 1 nm and 20 nm, the silica of which is bound with saponins and/or or the compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound is coupled to at least one saponin.
  • the functionalization may not be complete, ie the compounds thus functionalized may only partially cover said at least one magnetic compound.
  • the inorganic silicate compounds and all the compounds having an affinity for the at least magnetic compound and/or for the inorganic silicate compound listed above can be “functionalized” by a detergent of saponin type.
  • the inorganic silicate compound is a 7nm Ludox® silica nanoparticle and the compound having an affinity for the at least one magnetic compound and/or the at least one least one inorganic silicate compound is chosen from dopamine and its derivatives, catechols and their derivatives, phosphonic acids, phosphonates, phosphates and compounds of the carboxylic acid family (preferably citric acid and its salts), preferably coupled to at least one saponin.
  • the present invention relates to a molecular diagnostic kit comprising at least one three-layer complex according to the invention defined above.
  • the latter can also include reagents allowing the specific amplification of the nucleic acids likely or suspected to be contained in the sample to be tested, and/or reagents allowing the detection and/or the quantification of the nucleic acids likely or suspected of be contained in the sample to be tested
  • Example 1 Coating of magnetite particles with inorganic silicate compounds (magnetite/inorganic silicate compounds)
  • magnetite particles of about 100 nm, by commercial inorganic silicate compounds, were made with different magnetite/silica ratios.
  • Ludox SM ® bentonite, magnesium or sodium silicate, “smoke” silica or silicon dioxide.
  • Coating is favored by opposing electrostatic interactions.
  • the latter must be carried out in a pH zone where the inorganic silicate nanoparticles and the magnetite have opposite surface charges.
  • the measurement of the zeta potential of these particles as a function of the pH makes it possible to deduce the isoelectric point when the zeta potential becomes zero. It is indicative of the state of the surface functionalization of the magnetite.
  • Example 2 Coating of magnetite particles with compounds having an affinity for the at least one magnetic compound and/or for the at least one inorganic silicate compound.
  • these last compounds are also called ligands (magnetite/ligand)
  • Magnetite particles were coated with ligands as described below according to two protocols depending on the pKa of the compounds having an affinity for the at least one magnetic compound and/or for the at least one inorganic silicate compound (ligands) chosen.
  • an iron ligand will adsorb efficiently on magnetite in a pH range where the ligand is negatively charged and where the magnetite is positively charged either at a pH between the first pKa of the ligand and the isoelectric point of magnetite (6.8).
  • the coatings or coatings of compounds having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound of the phosphate type (pKa between 1 and 12) and phosphonates (pKa between 3 and 6) were carried out with respectively a pH of the solution equal to pH 3 or pH 5. It should be noted that any other compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound can be adsorbed (carboxylic acids, etc.) following this principle.
  • the table below contains a summary of a few examples of coating magnetite with compounds having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound (at a concentration optimum in ligand compound to satisfy the best conditions for nucleic acid extraction).
  • Example 3 Coating with compounds having an affinity for the at least one magnetic compound and/or for the at least one inorganic silicate compound of magnetite particles previously coated with inorganic silicate compounds (magnetite/Inorganic silicate compounds/ligands)
  • the magnetite/inorganic silicate compound composite particles obtained in example 1 are in turn coated with compounds having an affinity for the at least one magnetic compound and/or for the at least one inorganic silicate compound exactly as described in example 2 except that magnetite particles/silicated inorganic compounds are used instead of magnetite alone (see Table 3).
  • two coating protocols are used depending on the pKa of the compound having an affinity for the at least one magnetic compound and/or for the at least one inorganic silicate compound (ligand). Isoelectric point measurements are made on these particles.
  • Example 4 Measurements of DNA and RNA extraction yields with magnetic particles coated with silcate inorganic compounds described in example 1
  • the protocol described above makes it possible to measure a capture efficiency and an elution efficiency, based on what has been captured, as represented on the figure 4 and 5 . It is thus demonstrated that, compared to magnetite, the capture of DNA is only slightly modified by the coating of inorganic silicate compounds but that its elution is remarkably improved. On the other hand, there is no modification of the capture of the RNA and the coating of inorganic silicate compound also provides assistance in its elution (to a lesser extent). This phenomenon can be generalized to various types of inorganic silicate compounds as shown in the figures.
  • Example 5 Yield of extraction of DNA and RNA with magnetic particles coated with compounds having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound described in the example 2
  • Example 6 DNA and RNA Extraction Yield with Double Coated Magnetic Particles Described in Example 3
  • DNA and RNA extraction experiments were carried out following the protocol described in Example 4 using magnetic particles doubly coated: 1) with at least one inorganic silicate compound and 2) with at least a compound having an affinity for said at least one magnetic compound and/or for said at least one inorganic silicate compound as synthesized in example 3.
  • the double-coated particles give excellent results (represents the represented on the figure 9 and 10 ) and that they extract much better DNA and RNA than those that are coated once (particles of example 1 or 2), the extraction results are much better than the simple addition of the two extraction results with the two-layer complexes of examples 1 and 2, namely the magnetic particles coated with the inorganic silicate compound (example 1) and the particles coated with compounds having an affinity for the inorganic silicate compound and/or for the magnetic compound (example 2).
  • DNA capture capacity is substantially maintained compared to magnetite but elution is facilitated.
  • the capture capacity is also surprisingly maintained and the elution is very clearly favoured.
  • magnetite coated only with iron ligands gives very poor extraction results showing even more clearly the cooperative effect of the two types of coating (inorganic silicate compound + compound having an affinity for the at least one magnetic compound and/or the at least one inorganic silicate compound)
  • Example 8 Amplification and detection by real-time PCR of nucleic acids of the cytomegalo virus (CMV) extracted from a blood sample by tri-layer complexes according to the invention (particles as described in example 3).
  • CMV cytomegalo virus

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Claims (23)

  1. Dreischichtiger Komplex, umfassend:
    - eine erste Schicht, die mindestens eine magnetische Verbindung umfasst,
    - eine zweite Schicht, die die erste Schicht teilweise bedeckt und mindestens eine anorganische Silikatverbindung umfasst,
    - eine dritte Schicht, die die zweite Schicht zumindest teilweise bedeckt und mindestens eine Verbindung umfasst, die eine Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung besitzt, ausgewählt aus Zitronensäure und ihren Salzen, Phosphat-, Pyrophosphat-, Triphosphat-, Polyphosphationen, Phosphonsäuren, Phosphonaten, an organische Moleküle gekoppelten Phosphonaten oder Phosphonsäuren, Verbindungen aus der Familie der Phosphorsäuren, der Sulfonate, Verbindungen aus der Familie der Detergentien und/oder Verbindungen aus der Familie der Carbonsäuren.
  2. Dreischichtiger Komplex nach Anspruch 1, dadurch gekennzeichnet, dass die mindestens eine magnetische Verbindung aus Metallen und Metalloxiden ausgewählt ist.
  3. Dreischichtiger Komplex nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass die magnetische Verbindung aus Magnetit, Maghemit und Ferriten ausgewählt ist.
  4. Dreischichtiger Komplex nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die mindestens eine anorganische Silikatverbindung aus Silikaten, Magnesium- oder Natrium- oder Kalium- oder Lithium- oder Calciumsilikaten, Talk, Alumosilikaten, Kaolin, Bentonit, Siliziumdioxid-Nanopartikeln zwischen 0,1 und 20 nm, vorzugsweise zwischen 1 nm und 20 nm, mesoporösen Siliziumdioxid-Nanopartikeln, mit Siliziumdioxid bedeckten magnetischen Nanopartikeln, den vorstehend genannten Siliziumdioxid-Nanopartikeln, in denen das Siliziumdioxid an organische oder anorganische Gruppierungen gebunden ist, ausgewählt ist.
  5. Dreischichtiger Komplex nach Anspruch 4, dadurch gekennzeichnet, dass die mindestens eine anorganische Silikatverbindung aus Bentonit oder Siliziumdioxid-Nanopartikeln zwischen 0,1 nm und 20 nm, vorzugsweise zwischen 1 nm und 20 nm, besteht.
  6. Dreischichtiger Komplex nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die mindestens eine anorganische Silikatverbindung in partikulärer Form, wie ein Korn, ein Blättchen, eine Faser, eine Nadel, ein Nanopartikel..., vorzugsweise in nanopartikulärer Form, oder in einer in einem wässrigen Lösungsmittel, einem Gemisch von wässrigem Lösungsmittel/organischem Lösungsmittel oder einem organischen Lösungsmittel unlöslichen Form vorliegt.
  7. Dreischichtiger Komplex nach Anspruch 6, dadurch gekennzeichnet, dass die Partikel oder unlöslichen Formen der anorganischen Silikatverbindung eine Größe zwischen 0,1 und 20 nm, vorzugsweise zwischen 1 nm und 20 nm, bevorzugt zwischen 2 und 10 nm, noch stärker bevorzugt zwischen 6 und 8 nm aufweisen.
  8. Dreischichtiger Komplex nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die mindestens eine Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung organischer Natur ist.
  9. Dreischichtiger Komplex nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die mindestens eine Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung anorganischer Natur ist.
  10. Dreischichtiger Komplex nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die mindestens eine Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung aus Monophosphaten, Pyrophosphaten und/oder Triphosphaten besteht.
  11. Dreischichtiger Komplex nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass er außerdem einen festen Träger unter der gesamten oder einem Teil der ersten Schicht umfasst.
  12. Dreischichtiger Komplex nach Anspruch 11, dadurch gekennzeichnet, dass der Träger ein ebener Träger, ein Hohlträger, ein Wafer, eine Nadel, eine Membran, eine Platte, ein Kegel, eine Röhre, ein Kügelchen, ein Partikel, vorzugsweise ein Partikel, ist.
  13. Dreischichtiger Komplex nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass der Komplex in Partikelform vorliegt, und dadurch, dass sich die dritte Schicht auf der Außenseite des Partikels befindet.
  14. Komplex nach Anspruch 13, dadurch gekennzeichnet, dass die erste Schicht den Kern des Partikels bildet und eine Größe zwischen 2 und 400 nm, vorzugsweise zwischen 50 und 100 nm aufweist.
  15. Verfahren zur Herstellung mindestens eines dreischichtigen Komplexes nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass es mindestens die folgenden Schritte umfasst:
    a0) gegebenenfalls Inkontaktbringen eines Trägers nach Anspruch 12 mit mindestens einer magnetischen Verbindung nach Anspruch 2 oder 3, so dass sich die mindestens eine magnetische Verbindung am Träger festsetzt oder daran bindet,
    a) Inkontaktbringen des Ergebnisses von Schritt a0) oder mindestens einer magnetischen Verbindung nach Anspruch 2 oder 3 mit mindestens einer anorganischen Silikatverbindung nach einem der Ansprüche 4 bis 7, so dass eine elektrostatische Wechselwirkung und/oder eine kovalente Bindung und/oder eine koordinative Bindung zwischen der mindestens einen magnetischen Verbindung und der mindestens einen anorganischen Silikatverbindung auftritt und so dass die Schicht der mindestens einen anorganischen Silikatverbindung die Schicht der mindestens einen magnetischen Verbindung teilweise bedeckt, und
    b) Inkontaktbringen des Ergebnisses von Schritt a) mit mindestens einer Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung nach einem der Ansprüche 8 bis 10, vorzugsweise in wässrigem Medium, so dass die mindestens eine Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung über der Schicht der mindestens einen anorganischen Silikatverbindung des zweischichtigen Komplexes, mit oder ohne Träger, haftet und sich darüber positioniert.
  16. Verfahren zur Herstellung eines Komplexes nach einem der Ansprüche 1 bis 15, das mindestens die folgenden Schritte umfasst:
    a) Inkontaktbringen mindestens einer magnetischen Verbindung nach Anspruch 2 oder 3 mit mindestens einer anorganischen Silikatverbindung nach einem der Ansprüche 4 bis 7, wobei die anorganische Silikatverbindung bereits in partikulärer, nanopartikulärer oder unlöslicher Form vorliegt, so dass eine elektrostatische Wechselwirkung und/oder eine kovalente Bindung und/oder eine koordinative Bindung zwischen den mindestens zwei Verbindungen auftritt und so dass die Partikel oder unlöslichen Formen das den Kern bildende Partikel der mindestens einer magnetischen Verbindung teilweise bedecken, und
    b) Inkontaktbringen des nach Schritt a) erhaltenen umhüllten Partikels mit mindestens einer Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung nach einem der Ansprüche 8 bis 10, vorzugsweise in wässrigem Medium, so dass eine elektrostatische Wechselwirkung und/oder eine kovalente Bindung und/oder eine koordinative Bindung zwischen der mindestens einen Verbindung mit einer Affinität für die mindestens eine anorganische Silikatverbindung und/oder die mindestens eine magnetische Verbindung und der mindestens einen anorganischen Silikatverbindung des nach Schritt a) erhaltenen zweischichtigen Komplexes oder der mindestens einen magnetischen Verbindung des nach Schritt a) erhaltenen zweischichtigen Komplexes auftritt und so dass die mindestens eine Verbindung mit einer Affinität für die mindestens eine anorganische Silikatverbindung und/oder die mindestens eine magnetische Verbindung an der Oberfläche der mindestens einen anorganischen Silikatverbindung und/oder am Kern des endgültigen Partikels haftet.
  17. Verfahren zum Reinigen von Mikroorganismen und/oder Biomolekülen oder zur Extraktion von Biomolekülen, vorzugsweise Nukleinsäuren, aus einer Probe, bei dem man mindestens einen Komplex nach einem der Ansprüche 1 bis 14 oder erhalten nach einem der Ansprüche 15 oder 16 einsetzt.
  18. Verfahren zum Nachweis und/oder zur Quantifizierung von Zielnukleinsäuren aus einer Probe, die die Zielnukleinsäuren enthalten kann, das die folgenden Schritte umfasst:
    1. Extraktion der Nukleinsäuren aus einer Probe durch Einsatz des Verfahrens nach Anspruch 17,
    2. Nachweis und/oder zur Quantifizierung der Zielnukleinsäuren durch herkömmliche Nachweis- und/oder Quantifizierungstechniken.
  19. Verfahren zum Nachweis und/oder zur Quantifizierung von Zielnukleinsäuren nach Anspruch 18, dadurch gekennzeichnet, dass zwischen dem Schritt der Extraktion und dem Schritt des Nachweisens der Nukleinsäuren ein Schritt der Elution der Nukleinsäuren aus dem in Schritt 1) eingesetzten Komplex und/oder ein Schritt der Amplifikation der Nukleinsäuren durch herkömmliche Techniken erfolgt.
  20. Verfahren zur Lyse von Mikroorganismen und/oder Zellen und/oder Geweben aus einer Probe, dadurch gekennzeichnet, dass es aus dem Inkontaktbringen mindestens eine Probe mit mindestens einem Komplex nach einem der Ansprüche 1 bis 14 besteht und wobei die mindestens eine anorganische Silikatverbindung und/oder die mindestens eine Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung mindestens ein Mittel aus der Familie der Detergentien umfasst, das die Lyse ermöglicht.
  21. Verfahren nach Anspruch 20, wobei die mindestens eine anorganische Silikatverbindung Siliziumdioxid-Nanopartikel zwischen 0,1 nm und 20 nm, vorzugsweise zwischen 1 nm und 20 nm umfasst, worin das Siliziumdioxid an mindestens ein Saponin gebunden ist, und/oder die Verbindung mit einer Affinität für die mindestens eine magnetische Verbindung und/oder für die mindestens eine anorganische Silikatverbindung aus Dopamin und seinen Derivaten, Brenzkatechinen und ihren Derivaten, Phosphonsäuren, Phosphonaten, Phosphaten und Verbindungen aus der Familie der Carbonsäuren (vorzugsweise Zitronensäure und ihren Salzen), die an mindestens ein Saponin gekoppelt sind, ausgewählt ist.
  22. Molekulardiagnostik-Kit, das mindestens einen Komplex nach einem der Ansprüche 1 bis 14 umfasst.
  23. Molekulardiagnostik-Kit nach Anspruch 22, außerdem umfassend:
    - Reagenzien, die die spezifische Amplifikation von Nukleinsäuren ermöglichen, die in der zu testenden Probe enthalten sein können oder vermutlich enthalten sind, und/oder
    - Reagenzien, die den Nachweis und/oder die Quantifizierung der Nukleinsäuren ermöglichen, die in der zu testenden Probe enthalten sein können oder vermutlich enthalten sind.
EP15823699.2A 2014-12-30 2015-12-21 Mehrschichtiger komplex, verfahren zur herstellung dieses komplexes und verwendung dieses komplexes Active EP3240897B1 (de)

Applications Claiming Priority (2)

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FR1463423A FR3031063B1 (fr) 2014-12-30 2014-12-30 Complexe multicouches, procede de fabrication et utilisation du complexe
PCT/FR2015/053673 WO2016108004A1 (fr) 2014-12-30 2015-12-21 Complexe multicouches, procédé de fabrication et utilisation du complexe

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CN107250359B (zh) 2021-01-29
US10752892B2 (en) 2020-08-25
FR3031063A1 (fr) 2016-07-01
WO2016108004A1 (fr) 2016-07-07
PT3240897T (pt) 2022-05-18
ES2913199T3 (es) 2022-06-01
JP6815319B2 (ja) 2021-01-20
CN107250359A (zh) 2017-10-13
EP3240897A1 (de) 2017-11-08
FR3031063B1 (fr) 2017-02-10
PL3240897T3 (pl) 2022-11-07
JP2018512111A (ja) 2018-05-17
US20170342400A1 (en) 2017-11-30

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